TRIM5alpha proteins bind retroviral capsids after cell entry and restrict retroviral infection by blocking reverse transcription and/or integration of the viral genetic material {Nisole, 2005 #305;Towers, 2007 #310}. This novel mechanism of cellular immunity against retroviruses appears to determine the species tropism of the primate immunodeficiency viruses active today. Experimental evidence suggests that species-specific differences in TRIM5alpha activity arise from differences in TRIM5alpha affinity for the capsid. Capsid recognition is mediated by the B30.2 domain of TRIM5alpha, but the structural basis of TRIM5alpha-CA interactions is unknown. The central hypothesis of this proposal is that structural differences at the B30.2- capsid interface explain species-specific differences in TRIM5alpha activity. In order to elucidate the mechanism of the TRIM5alpha-capsid binding I will pursue the following objectives: 1. TRIM5alpha B30.2 domain structure: Expression and purification protocols will be developed in order to produce and label TRIM5alpha proteins in sufficient quantities for structural and biophysical studies. Structures of the rhesus and human TRIM5alpha B30.2 domains will be determined using NMR. Effects of the TRIM5alpha specificity-altering mutations on B30.2 structure will be investigated. Structural differences between rhB30.2 and huB30.2 responsible for the inability of the huTRIM5alpha to restrict HIV will be determined. Dynamic parameters of the B30.2 variable loops involved in capsid binding will be measured. 2. Molecular basis of capsid recognition by TRIM5alpha: Interaction surfaces involved in B30.2-capsid binding will be identified using NMR. Relative orientation of B30.2 and CA in the complex will be determined. A model of the B30.2-CA complex will be produced and key B30.2-CA interactions identified. Structural models will be tested using mutagenesis, biophysical and in-vivo assays. NMR will be used to detect cooperativity between CypA-CA and TRIM5alpha-CA binding and to check whether CypA-catalyzed cis-trans isomerisation of the G89-P90 peptide bond of the HIV-1 capsid affects B30.2-CA interactions. PUBLIC HEALTH RELEVANCE: The AIDS epidemic caused by the HIV retrovirus is one of the leading threats posed to global health by an infectious agent. Higher organisms have multiple layers of immunity against retroviral pathogens, but the HIV has evolved specific mechanisms to overcome host defenses. The goal of this proposal is to investigate the mechanism of retroviral restriction by the primate TRIM5alpha proteins, to elucidate viral evasion strategies and to explore whether the inability of the human TRIM5alpha to restrict HIV could be restored by pharmacological means.